1. Scope of the Invention
This invention relates to a method for enhancing the anti-infective activity of muramyldipeptides. More specifically the anti-infective activity of muramyldipeptides may be enhanced by the serial administration of these compounds with liposomes.
1. Background of the Invention
In the field of immunology, more than one injection of a vaccine (or bacterin) is frequently required to achieve the immunological response in a host sufficient to ward off an infection. During the development of the vaccine art it was discovered that substances could be added to the vaccine composition which would enhance the antigenicity of the vaccine to give a superior immune response over that achieved with injection of the vaccine alone. Among the most effective adjuvants developed early on in the vaccine art was Freund's complete adjuvant which is a suspension of killed, whole Mycobacterium tuberculosis in mineral oil plus an emulsifier. Also highly effective was Freund's incomplete adjuvant which is a mineral oil plus an emulsifier only. Notwithstanding that these two adjuvants were used as laboratory standards, they were and are not commercially used because Freund's complete adjuvant is derived from a virulent microorganism, Mycrobacterium tuberculosis, and both the complete and incomplete forms contain mineral oil, a human carcinogen. Also, in the absence of the microorganism, this adjuvant does not always produce a satisfactorily high immunological enhancement.
Attempts to find the basic unit responsible for enhancing the cellular antigenicity response resulted in the finding that sugar-containing peptides of the cell wall were the immune stimulating component in the bacterial extracts. A detailed study of the chemistry of mycobacterial cell wall resulted in the observation by F. Ellouz, et al, Biochem Biophys, Res. Comm. 59, 1317 (1974) that the adjuvant activity could be directly attributable to the bacterial wall peptidoglycan derivatives. The smallest effective molecule was found to be an N-acetylmuramyldipeptide, specifically N-acetylmuramyl-L-alanyl-D-isoglutamine, prepared by C. Messer, P. Sinay, and A. Adams, Biochem, Biophys Res. Comm. 66, 1316 (1975). This compound is now commonly called muramyldipeptide or MDP.
Subsequently a number of muramyldipeptide analogs and derivatives have been prepared by various academic and industrial concerns. The majority of these compounds have, to one degree or another, been demonstrated to be effective adjuvants. In addition, many of these muramyldipeptide analogs and derivatives are, per se, active against infectious organisms such as Klebsiella pneumoniae. Escherichia coli, Candida albicans, Staphylococcus aureus and the like. The journal literature on MDP compounds and their immunological activities is extensive. A number of patents have issued on many such compounds and patent applications are pending on others.
The materials responsible for enhancing the anti-infective activity of muramyldipeptides are liposomes. They are microscopic vesicles, generally spherically shaped, formed from one or several concentric layers (lamellae) of lipid molecules having a lipophilic and hydrophilic moiety. Most frequently, liposomes are water insoluble, amphapathic phospholipids which form bilayer structures spontaneously in aqueous solution. Regardless of the overall shape, the bilayers are organized as closed concentric lamella, with an aqueous layer separating each lamella from its neighbor. The lamella of water soluble liposomes comprise at least one bilipid layer, the molecules of this layer being so oriented that the hydrophilic functions are in contact with the aqueous phase. Since the liposomes lamella layers are being separated from each other by a water film, they have a wall-like structure which can be schematically represented, in sections, by molecular composite XY-YX, X representing the hydrophilic portion of the molecule and Y the lipophilic portion. Liposome vesicle size is highly variable and dependent on the method of manufacture thereof; but generally they have a 25 to 30,000 nm diameter and the film between the bilayer of 3 to 10 nm.
In recent years liposomes have attracted widespread interest from a variety researchers for a number of reasons ranging from the purely theoretical physical chemistry point of view to projected applications, particularly in medicine.
The physical chemistry studies have focused on such properties as fluidity, permeability, and molecular organization. These studies are generally motivated by the importance of the lipid bilayer as a structural element of natural membranes. Liposomes, it has been found, can be used to promote cell cell fussion, alter membrane phospholipid and cholesterol content, and transfer water-soluble, normally impermenant molecules into cells.
In clinical drug research, liposomes have been viewed as a pharmaceutical capsule for the possible selective delivery of therapeutic agents such as insulin, enzymes, interferon and other anti-tumor drugs.
Liposome-encapsulated muramyldipeptide, the native compound, has been used in studies wherein noncytotoxic macrophages have been rendered tumoricidal by the interaction of macrophage-activating factor and free or encapsulated MDP in studies reported by S. Sone and I. J. Fidler, The Journal of Immunology, vol. 125, number 6, pp. 2454-2460 (1980). Two subsequent papers which describe continued studies in this area by are Sone and Fidler (Cellular Immunology, 57, 42-50 (1981)) and I. J. Fidler, et al (Proc. Natl. Acad. Sci. USA, 78, 1680-1684 (1981)). The in vivo studies of Fidler, et al reported in PNAS indicate that empty multi-lameler vesicles plus free MDP did not activate tumoricidal activity in mice alveolar macrophages when free MDP was administered at the same level as that adequate to cause activation by liposome-encapsulated MDP. These investigators found that a dose of MDP 80 times greater than the liposome encapsulated MDP dose did not activate the tumoricidal activity of mice alveolar macrophages.
It has been shown that the dose of an MDP derivative required for efficacy in protecting mice from bacterial or yeast infections can be reduced significantly by encapsulating the analog in liposomes. For example, to achieve protection against Candida albicans yeast infection in mice, the MDP derivative dose was reduced by approximately 15-fold by its encapsulation with multilamellar liposome vesicles, efficacy being also related to the liposome composition. This increased efficacy was only achieved when the liposome-encapsulated drug was given by intravenous injection, which allowed the liposomes to be targeted to the phagocytic cells of the reticuloendothelial system. (See E. B. Fraser-Smith, et al, ASM meeting, March 1982.)
However, it has now been determined that this same increased efficacy can also be achieved without actual encapsulation of the drug in liposomes, or without other types of physical association of the drug with liposomes (such as association with the lipid bilayers) but by simply co-administering the MDP analog with liposomes. The drug can either be mixed with the preformed liposomes prior to injection, or the drug can be injected first followed by injection of the liposomes or liposomes can be injected first, followed by injection of the drug. Such serial administration of liposomes and free MDP compound provides essentially the same protective activity as obtained with the same dose of liposome-encapsulated MDP compound whereas the MDP compound alone was ineffective at a similar concentration and was not found to impart protective activity on an equivalent basis until the dose was increased by at least an order of magnitude.
Therefore, the central aspect of this invention relates to a novel method for enhancing the anti-infective activity of native muramyldipeptide or its analogs and derivatives by serial injection of such compound with liposomes.